Methods of using cyanovirins to inhibit viral infection

ABSTRACT

The present invention provides antiviral proteins, peptides and conjugates, as well as methods of obtaining these agents. The antiviral proteins, peptides and conjugates of the present invention can be used alone or in combination with other antiviral agents in compositions, such as pharmaceutical compositions, to inhibit the infectivity, replication and cytopathic effects of a virus, such as a retrovirus, in particular a human immunodeficiency virus, specifically HIV-1 or HIV-2, in the treatment or prevention of viral infection.

This is a continuation Ser. No. 08/969,378, filed on Nov. 13, 1997 nowU.S. Pat. No. 6,015,876, which is a divisional of Ser. No. 08/429,965,filed on Apr. 27, 1995, U.S. Pat. No. 5,843,882.

TECHNICAL FIELD OF THE INVENTION

This invention relates to antiviral proteins and peptides, collectivelyreferred to as cyanovirins, and conjugates thereof, as well as methodsof obtaining antiviral cyanovirins and conjugates thereof, compositionscomprising cyanovirins and conjugates thereof, and methods of usingcyanovirins and conjugates thereof in clinical applications, such as inantiviral therapy and prophylaxis.

BACKGROUND OF THE INVENTION

Acquired immune deficiency syndrome (AIDS) is a fatal disease, reportedcases of which have increased dramatically within the past severalyears. The AIDS virus was first identified in 1983. It has been known byseveral names and acronyms. It is the third known T-lymphotropic virus(HTLV-III), and it has the capacity to replicate within cells of theimmune system, causing profound cell destruction. The AIDS virus is aretrovirus, a virus that uses reverse transcriptase during replication.This particular retrovirus is also known as lymphadenopathy-associatedvirus (LAV), AIDS-related virus (ARV) and, most recently, as humanimmunodeficiency virus (HIV). Two distinct families of HIV have beendescribed to date, namely HIV-1 and HIV-2. The acronym HIV is usedherein to refer to human immunodeficiency viruses generically.

HIV exerts profound cytopathic effects on the CD4⁺ helper/inducerT-cells, thereby severely compromising the immune system. HIV infectionalso results in neurological deterioration and, ultimately, in death ofinfected individuals. Tens of millions of people are infected with HIVworldwide, and, without effective therapy, most of these are doomed todie. During the long latency, the period of time from initial infectionto the appearance of symptoms, or death, due to AIDS, infectedindividuals spread the infection further, by sexual contacts, exchangesof contaminated needles during i.v. drug abuse, transfusions of blood orblood products, or maternal transfer of HIV to a fetus or newborn. Thus,there is not only an urgent need for effective therapeutic agents toinhibit the progression of HIV disease in individuals already infected,but also for methods of prevention of the spread of HIV infection frominfected individuals to noninfected individuals. Indeed, the WorldHealth Organization (WHO) has assigned an urgent international priorityto the search for an effective anti-HIV prophylactic virucide to helpcurb the further expansion of the AIDS pandemic (Balter, Science 266,1312-1313, 1994; Merson, Science 260, 1266-1268, 1993; Taylor, J. NIHRes. 6, 26-27, 1994; Rosenberg et al., Sex. Transm. Dis. 20, 41-44,1993; and Rosenberg, Am. J. Public Health 82, 1473-1478, 1992).

The field of viral therapeutics has developed in response to the needfor agents effective against retroviruses, especially HIV. There aremany ways in which an agent can exhibit anti-retroviral activity (e.g.,see DeClercq, Adv. Virus Res. 42, 1-55, 1993; DeClercq, J. Acquir.Immun. Def. Synd. 4, 207-218, 1991; and Mitsuya et al., Science 249,1533-1544, 1990). Nucleoside derivatives, such as AZT, which inhibit theviral reverse transcriptase, are the only clinically active agents thatare currently available commercially for anti-HIV therapy. Although veryuseful in some patients, the utility of AZT and related compounds islimited by toxicity and insufficient therapeutic indices for fullyadequate therapy. Also, given the recent revelations about the truedynamics of HIV infection (Coffin, Science 267, 483-489, 1995; andCohen, Science 267, 179, 1995), it is now increasingly apparent thatagents acting as early as possible in the viral replicative cycle areneeded to inhibit infection of newly produced, uninfected immune cellsgenerated in the body in response to the virus-induced killing ofinfected cells. Also, it is essential to neutralize or inhibit newinfectious virus produced by infected cells.

Therefore, new classes of antiviral agents, to be used alone or incombination with AZT and/or other available antiviral agents, are neededfor effective antiviral therapy against AIDS. New agents, which may beused to prevent HIV infection, are also important for prophylaxis. Inboth areas of need, the ideal new agent(s) would act as early aspossible in the viral life cycle; be as virus-specific as possible(i.e., attack a molecular target specific to the virus but not thehost); render the intact virus noninfectious; prevent the death ordysfunction of virus-infected cells; prevent further production of virusfrom infected cells; prevent spread of virus infection to uninfectedcells; be highly potent and active against the broadest possible rangeof strains and isolates of HIV; be resistant to degradation underphysiological and rigorous environmental conditions; and be readily andinexpensively produced on a large-scale basis.

Accordingly, it is an object of the present invention to provideantiviral proteins and peptides, and conjugates thereof, which possessthe aforementioned particularly advantageous attributes.

It is a related object of the present invention to provide conjugates orchimeras containing an antiviral protein or peptide coupled to aneffector molecule.

It is still another object of the present invention to provide acomposition, in particular a pharmaceutical composition, which inhibitsthe growth or replication of a virus, such as a retrovirus, inparticular a human immunodeficiency virus, specifically HIV-1 or HIV-2.

It is another object of the present invention to provide methods ofobtaining an antiviral protein or peptide or conjugate thereof.

It is yet another object of the present invention to provide nucleicacid molecules, including recombinant vectors, encoding such antiviralproteins and peptides and conjugates thereof. A more specific object ofthe present invention is to provide a DNA coding sequence comprising SEQID NO:1.

It is another specific object of the present invention to provide a DNAcoding sequence comprising SEQ ID NO:3.

Yet another object of the present invention is to provide a method ofusing an antiviral protein or peptide to target an effector molecule tovirus and/or to virus-producing cells, specifically to retrovirus and/orto retrovirus-producing cells, more specifically to HIV and/orHIV-producing cells, and even more specifically to viral gp120 and/orcell-expressed gp120.

Still yet another object of the present invention is to provide a methodof treating an animal, in particular a human, infected by a virus, suchas a retrovirus, in particular a human immunodeficiency virus,specifically HIV-2 or HIV-2. A related object of the present inventionis to provide a method of treating an animal, in particular a human, toprevent infection by a virus, such as a retrovirus, in particular ahuman immunodeficiency virus, specifically HIV-1 or HIV-2.

It is another related object of the present invention to provide amethod of treating inanimate objects, such as medical and laboratoryequipment and supplies, to prevent infection of an animal, in particulara human, by a virus, such as a retrovirus, in particular a humanimmunodeficiency virus, specifically HIV-1 or HIV-2. It is a furtherrelated object of the present invention to provide a method of treatinginjectable or infusible fluids, suspensions or solutions, such as bloodor blood products, and tissues to prevent infection of an animal, inparticular a human, by a virus, such as a retrovirus, in particular ahuman immunodeficiency virus, specifically HIV-1 or HIV-2.

These and other objects of the present invention, as well as additionalinventive features, will become apparent from the description providedherein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antiviral agents, in particular antiviralproteins and peptides, collectively referred to as cyanovirins, andconjugates thereof, which are useful for antiviral therapy andprophylaxis. Cyanovirins and conjugates thereof inhibit the infectivity,cytopathicity and replication of a virus, in particular a retrovirusspecifically a human immunodeficiency virus, such as HIV-1 or HIV-2.Also provided are methods of obtaining a cyanovirin and a conjugatethereof. Nucleic acid molecules, including nucleic acid molecules ofspecified nucleotide sequence and recombinant vectors, encodingcyanovirins and conjugates thereof are also provided. The invention alsoprovides a method of using a cyanovirin to target an effector moleculeto a virus, such as a retrovirus, specifically HIV, and/or avirus-producing, such as a retrovirus-producing, specificallyHIV-producing, cell, in particular viral gp120 and/or cell-expressgp120. The present invention also provides a method of obtaining asubstantially pure cyanovirin and a conjugate thereof. The cyanovirin orconjugate thereof can be used in a composition, such as a pharmaceuticalcomposition, which can additionally comprise one or more other antiviralagents. The cyanovirin, conjugate, and composition thereof, alone or incombination with another antiviral agent, therefore, is useful in thetherapeutic and prophylactic treatment of an animal, such as a human,infected or at risk for infection with a virus, particularly aretrovirus, specifically a human immunodeficiency virus, such as HIV-1or HIV-2, and in the treatment of inanimate objects, such as medical andlaboratory equipment and supplies, suspensions or solutions, such asblood and blood products, and tissues to prevent viral infection of ananimal, such as a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of OD 206 nm versus time (min), which represents anHPLC chromatogram of nonreduced cyanovirin.

FIG. 1B is a bar graph of maximum dilution for 50% protection versusHPLC fraction, which illustrates the maximum dilution of each HPLCfraction that provided 50% protection from the cytopathic effects of HIVinfection for the nonreduced cyanovirin HPLC fractions.

FIG. 1C is a graph of OD 206 nm versus time (min), which represents anHPLC chromatogram of reduced cyanovirin.

FIG. 1D is a bar graph of maximum dilution for 50% protection versusHPLC dilution, which illustrates the maximum dilution of each fractionthat provided 50% protection from the cytopathic effects of HIVinfection for the reduced cyanovirin HPLC fractions.

FIG. 2 shows an example of a DNA sequence encoding a syntheticcyanovirin gene SEQ ID NOS: 1-4.

FIG. 3 illustrates a site-directed mutagenesis maneuver used toeliminate codons for a FLAG octapeptide and a Hind III restriction sitefrom the sequence of FIG. 2.

FIG. 4 shows a typical HPLC chromatogram during the purification of arecombinant native cyanovirin.

FIG. 5A is a graph of % control versus concentration (nm), whichillustrates the antiviral activity of native cyanovirin from Nostocellipsosporum.

FIG. 5B is a graph of % control versus concentration (nm), whichillustrates the antiviral activity of recombinant cyanovirin.

FIG. 5C is a graph of % control versus concentration (nm), whichillustrates the antiviral activity of recombinant FLAG-fusioncyanovirin.

FIG. 6A is a graph of % control versus concentration (nm), which depictsthe relative numbers of viable CEM-SS cells infected with HIV-1 in aBCECF assay.

FIG. 6B is a graph of % control versus concentration (nm), which depictsthe relative, DNA contents of CEM-SS cell cultures infected with HIV-1.

FIG. 6C is a graph of % control versus concentration (nm), which depictsthe relative numbers of viable CEM-SS cells infected with HIV-1 in anXTT assay.

FIG. 6D is a graph of % control versus concentration (nm), which depictsthe effect of a range of concentration of cyanovirin upon indices ofinfectious virus or viral replication.

FIG. 7A is a graph of % uninfected control versus time of addition(hrs), which shows results of time-of-addition studies of a cyanovirin,showing anti-HIV activity in CEM-SS cells infected with HIV-1_(RF).

FIG. 7B is a bar graph of % control versus time of addition (hrs) versus% control RT (reverse transcriptase).

FIG. 8 is a graph of OD (450 nm) versus cyanovirin concentration(μg/ml), which illustrates cyanovirin/gp120 interactions defining gp120as a principal molecular target of cyanovirin.

FIG. 9 is a flowchart of the synthesis of the DNA sequence as describedin Example 2.

FIG. 10 is a flowchart of the synthesis of the expression of syntheticcyanovirin genes as described in Example 3.

FIG. 11 is a flowchart of the purification of recombinant cyanovirinproteins as described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Infection of CD4⁺ cells by HIV-1 and related primate immunodeficiencyviruses begins with interaction of the respective viral envelopeglycoproteins (generically termed “gp120”) with the cell-surfacereceptor CD4, followed by fusion and entry (Sattentau, AIDS 2, 101-105,1988; and Koenig et al., PNAS USA 86, 2443-2447, 1989). Productivelyinfected, virus-producing cells express gp120 at the cell surface;interaction of gp120 of infected cells with CD4 on uninfected cellsresults in formation of dysfunctional multicellular syncytia and furtherspread of viral infection (Freed et al., Bull. Inst. Pasteur 88, 73,p990). Thus, the gp120/CD4 interaction is a particularly attractivetarget for interruption of HIV infection and cytopathogenesis, either byprevention of initial virus-to-cell binding or by blockage ofcell-to-cell fusion (Capon et al., Ann. Rev. Immunol. 9, 649-678, 1991).Virus-free or “soluble” gp120 shed from virus or from infected cells invivo is also an important therapeutic target, since it may otherwisecontribute to noninfectious immunopathogenic processes throughout thebody, including the central nervous system (Capon et al., 1991, supra;and Lipton, Nature 367, 113-114, 1994). Much vaccine research hasfocused upon gp120; however, progress has been hampered byhypervariability of the gp120-neutralizing determinants, and consequentextreme strain-dependence of viral sensitivity to gp120-directedantibodies (Berzofsky, J. Acq. Immun. Def. Synd. 4, 451-459, 1991).Relatively little drug discovery and development research has focusedspecifically upon gp120. A notable exception is the considerable effortthat has been devoted to truncated, recombinant “CD4” proteins (“solubleCD4” or “sCD4”), which bind gp120 and inhibit HIV infectivity in vitro(Capon et al., 1991, supra; Schooley et al., Ann. Int. Med. 112,247-253, 1990; and Husson et al., J. Pediatr. 121, 627-633, 1992).However, clinical isolates, in contrast to laboratory strains of HIV,have proven highly resistant to neutralization by sCD4 (Orloff et al.,AIDS Res. Hum. Retrovir. 11, 335-342, 1995; and Moore et al., J. Virol.66, 235-243, 1992). Initial clinical trials of sCD4 (Schooley et al.,1990, supra; and Husson et al., 1992, supra), and of sCD4-coupledimmunoglobulins (Langner et al., Arch. Virol. 130, 157-170, 1993), andlikewise of sCD4-coupled toxins designed to bind and destroyvirus-expressing cells (Davey et al., J. Infect. Dis. 170, 1180-1188,1994; and Ramachandran et al., J. Infect. Dis. 170, 1009-1113, 1994),have been disappointing. Newer gene-therapy approaches to generatingsCD4 directly in vivo (Morgan et al., AIDS Res. Hum. Retrovir. 10,1507-1515, 1994) will likely suffer similar frustrations.

In view of the above, the principal overall objective of the presentinvention is to provide anti-viral proteins, peptides and derivativesthereof, and broad medical uses thereof, including prophylactic and/ortherapeutic applications against viruses, such as retroviruses, inparticular a human immunodeficiency virus, specifically HIV-1 or HIV-2.

An initial observation, which led to the present invention, wasantiviral activity in certain extracts from cultured cyanobacteria(blue-green algae) tested in an anti-HIV screen. The screen is one thatwas conceived in 1986 (by M. R. Boyd of the National Institutes ofHealth) and has been developed and operated at the U.S. National CancerInstitute (NCI) since 1988 (see Boyd, in AIDS, Etiology, Diagnosis,Treatment and Prevention, DeVita et al., eds., Philadelphia: Lippincott,1988, pp. 305-317).

Cyanobacteria (blue-green algae) were specifically chosen for anti-HIVscreening because they had been known to produce a wide variety ofstructurally unique and biologically active non-nitrogenous and aminoacid-derived natural products (Faulkner, Nat. Prod. Rep. 11, 355-394,1994; and Glombitza et al., in Algal and Cyanobacterial Biotechnoloqy,Cresswell, R. C., et al. eds., 1989, pp. 211-218). These photosyntheticprocaryotic organisms are significant producers of cyclic and linearpeptides (molecular weight generally <3 kDa), which often exhibithepatotoxic or antimicrobial properties (Okino et al., Tetrahedron Lett.34, 501-504, 1993; Krishnamurthy et al., PNAS USA 86, 770-774, 1989;Sivonen et al., Chem. Res. Toxicol. 5, 464-469, 1992; Carter et al., J.Org. Chem. 49, 236-241, 1984; and Frankmolle et al., J. Antibiot. 45,1451-1457, 1992). Sequencing studies of higher molecular weightcyanobacterial peptides and proteins have generally focused on thoseassociated with primary metabolic processes or ones that can serve asphylogenetic markers (Suter et al., FEBS Lett. 217, 279-282, 1987;Rumbeli et al., FEBS Lett. 221, 1-2, 1987; Swanson et al., J. Biol.Chem. 267, 16146-16154, 1992; Michalowski et al., Nucleic Acids Res. 18,2186, 1990; Sherman et al., in The Cyanobacteria, Fay et al., eds.,Elsevier: New York, 1987, pp. 1-33; and Rogers, in The Cyanobacteria,Fay et al., eds., Elsevier: New York, 1987, pp. 35-67). In general,proteins with antiviral properties have not been associated withcyanobacterial sources.

The cyanobacterial extract leading to the present invention was amongmany thousands of different extracts initially selected randomly andtested blindly in the anti-HIV screen described above. A number of theseextracts. had been determined preliminarily to show anti-HIV activity inthe NCI screen (Patterson et al., J. Phycol. 29, 125-130, 1993). Fromthis group, an aqueous extract from Nostoc ellipsosporum, which had beenprepared as described (Patterson, 1993, supra) and which showed anunusually high anti-HIV potency and in vitro “therapeutic index” in theNCI primary screen, was selected for detailed investigation. A specificbioassay-guided strategy was used to isolate and purify a homogenousprotein highly active against HIV.

In the bioassay-guided strategy, initial selection of the extract forfractionation, as well as the decisions concerning the overall chemicalisolation method to be applied, and the nature of the individual stepstherein, were determined by interpretation of biological testing data.The anti-HIV screening assay (e.g., see Boyd, 1988, supra; Weislow etal., J. Natl. Cancer Inst. 81, 577-586, 1989), which was used to guidethe isolation and purification process, measures the degree ofprotection of human T-lymphoblastoid cells from the cytopathic effectsof HIV. Fractions of the extract of interest are prepared using avariety of chemical means and are tested blindly in the primary screen.Active fractions are separated further, and the resulting subfractionsare likewise tested blindly in the screen. This process is repeated asmany times as necessary in order to obtain the active compound(s), i.e.,antiviral fraction(s) representing pure compound(s), which then can besubjected to detailed chemical analysis and structural elucidation.

Using this strategy, aqueous extracts of Nostoc ellipsosporum were shownto contain an antiviral protein. Accordingly, the present inventionprovides an isolated and purified antiviral protein, named cyanovirin-N,from Nostoc ellipsosporum. Herein the term “cyanovirin” is usedgenerically to refer to a native cyanovirin or any related, functionallyequivalent protein, peptide or derivative thereof. By definition, inthis context, a related, functionally equivalent protein, peptide orderivative thereof a) contains a sequence of at least nine amino acidsdirectly homologous with any subsequence of nine contiguous amino acidscontained within a native cyanovirin, and, b) is capable of specificallybinding to virus, more specifically a primate immunodeficiency virus,more specifically HIV-1, HIV-2 or SIV, or to an infected host cellexpressing one or more viral antigen(s), more specifically an envelopeglycoprotein, such as gp120, of the respective virus. Herein, the term“protein” refers to a sequence comprising 100 or more amino acids,whereas “peptide” refers to a sequence comprising less than 100 aminoacids. Preferably, the protein, peptide or derivative thereof comprisesan amino acid sequence that is substantially homologous to that of anantiviral protein from Nostoc ellipsosporum. By “substantiallyhomologous” is meant sufficient homology to render the protein, peptideor derivative thereof antiviral, with antiviral activity characteristicof an antiviral protein isolated from Nostoc ellipsosporum. At leastabout 50% homology, preferably at least about 75% homology, and mostpreferably at least about 90% homology should exist. A cyanovirinconjugate comprises a cyanovirin coupled to one or more selectedeffector molecule(s), such as a toxin or immunological reagent.“Immunological reagent” will be used to refer to an antibody, animmunoglobulin, and an immunological recognition element. Animmunological recognition element is an element, such as a peptide,e.g., the FLAG sequence of the recombinant cyanovirin-FLAG fusionprotein, which facilitates, through immunological recognition, isolationand/or purification and/or analysis of the protein or peptide to whichit is attached. A cyanovirin fusion protein is a type of cyanovirinconjugate, wherein a cyanovirin is coupled to one or more otherprotein(s) having any desired properties or effector functions, such ascytotoxic or immunological properties, or other desired properties, suchas to facilitate isolation, purification or analysis of the fusionprotein.

Accordingly, the present invention provides an isolated and purifiedprotein encoded by a nucleic acid molecule comprising a sequence of SEQID NO:1, a nucleic acid molecule comprising a sequence of SEQ ID NO:3, anucleic acid molecule encoding an amino acid sequence of SEQ ID NO:2, ora nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:4.Preferably, the aforementioned nucleic acid molecules encode at leastnine contiguous amino acids of the amino acid sequence of SEQ ID NO: 2.

The present invention also provides a method of obtaining a cyanovirinfrom Nostoc ellipsosporum. Such a method comprises (a) identifying anextract of Nostoc ellipsosporum containing antiviral activity, (b)optionally removing high molecular weight biopolymers from the extract,(c) antiviral bioassay-guided fractionating the extract to obtain acrude extract of cyanovirin, and (d) purifying the crude extract byreverse-phase HPLC to obtain cyanovirin (see, also, Example 1). Morespecifically, the method involves the use of ethanol to remove highmolecular weight biopolymers from the extract and the use of an anti-HIVbioassay to guide fractionation of the extract.

Cyanovirin-N, which was isolated and purified using the aforementionedmethod, was subjected to conventional procedures typically used todetermine the amino acid sequence of a given pure protein. Thus, thecyanovirin was initially sequenced by N-terminal Edman degradation ofintact protein and numerous overlapping peptide fragments generated byendoproteinase digestion. Amino acid analysis was in agreement with thededuced sequence. ESI mass spectrometry of reduced, HPLC-purifiedcyanovirin-N showed a molecular ion consistent with the calculatedvalue. These studies indicated that cyanovirin-N from Nostocellipsosporum was comprised of a unique sequence of 101 amino acidshaving little or no significant homology to previously describedproteins or transcription products of known nucleotide sequences. Nomore than eight contiguous amino acids from cyanovirin were found in anyamino acid sequences from known proteins, nor were there any knownproteins from any source containing greater than 13% sequence homologywith cyanovirin-N. Given the chemically deduced amino acid sequence ofcyanovirin-N, a corresponding recombinant cyanovirin-N (r-cyanovirin-N)was created and used to definitively establish that the deduced aminoacid sequence was, indeed, active against virus, such as HIV (Boyd etal., 1995, supra; also, see Examples 2-5).

Accordingly, the present invention provides isolated and purifiednucleic acid molecules and synthetic nucleic acid molecules, whichcomprise a coding sequence for a cyanovirin, such as an isolated andpurified nucleic acid molecule comprising a sequence of SEQ ID NO:1, anisolated and purified nucleic acid molecule comprising a sequence of SEQID NO:3, an isolated and purified nucleic acid molecule encoding anamino acid sequence of SEQ ID NO:2, an isolated and purified nucleicacid molecule encoding an amino acid sequence of SEQ ID NO:4, and anucleic acid molecule that is substantially homologous to any one ormore of the aforementioned nucleic acid molecules. By “substantiallyhomologous” is meant sufficient homology to render the protein, peptideor derivative thereof antiviral, with antiviral activity characteristicof an antiviral protein isolated from Nostoc ellipsosporum. At leastabout 50% homology, preferably at least about 75% homology, and mostpreferably at least about 90% homology should exist. More specifically,the present invention provides one of the aforementioned nucleic acidmolecules, which comprises a nucleic acid sequence encoding at leastnine contiguous amino acids of the amino acid sequence of SEQ ID NO:2.

Given the present disclosure, it will be apparent to one skilled in theart that a partial cyanovirin-N gene codon sequence will likely sufficeto code for a fully functional, i.e., antiviral, such as anti-HIV,cyanovirin. A minimum essential DNA coding sequence(s) for a functionalcyanovirin can readily be determined by one skilled in the art, forexample, by synthesis and evaluation of sub-sequences comprising thenative cyanovirin, and by site-directed mutagenesis studies of thecyanovirin-N DNA coding sequence.

Using an appropriate DNA coding sequence, a recombinant cyanovirin canbe made by genetic engineering techniques (for general background see,e.g., Nicholl, in An Introduction to Genetic Engineering, CambridgeUniversity Press: Cambridge, 1994, pp. 1-5 & 127-130; Steinberg et al.,in Recombinant DNA Technoloqy Concepts and Biomedical Applications,Prentice Hall: Englewood Cliffs, N.J., 1993, pp. 81-124 & 150-162; Soferin Introduction to Genetic Engineering, Butterworth-Heinemann, Stoneham,Mass., 1991, pp. 1-21 & 103-126; Old et al., in Principles of GeneManipulation, Blackwell Scientific Publishers: London, 1992, pp. 1-13 &108-221; and Emtage, in Delivery Systems for Peptide Drugs, Davis etal., eds., Plenum Press: New York, 1986, pp. 23-33). For example, aNostoc ellipsosporum gene or cDNA encoding a cyanovirin can beidentified and subcloned. The gene or cDNA can then be incorporated intoan appropriate expression vector and delivered into an appropriateprotein-synthesizing organism (e.g., E. coli, S. cerevisiae, P.pastoris, or other bacterial, yeast, insect or mammalian cells, wherethe gene, under the control of an endogenous or exogenous promoter, canbe appropriately transcribed and translated. Such expression vectors(including, but not limited to, phage, cosmid, viral, and plasmidvectors) are known to those skilled in the art, as are reagents andtechniques appropriate for gene transfer (e.g., transfection,electroporation, transduction, micro-injection, transformation, etc.).Subsequently, the recombinantly produced protein can be isolated andpurified using standard techniques known in the art (e.g.,chromatography, centrifugation, differential solubility, isoelectricfocusing, etc.), and assayed for antiviral activity.

Alternatively, a native cyanovirin can be obtained from Nostocellipsosporum by non-recombinant methods (e.g., see Example 1 andabove), and sequenced by conventional techniques. The sequence can thenbe used to synthesize the corresponding DNA, which can be subcloned intoan appropriate expression vector and delivered into a protein-producingcell for en mass recombinant production of the desired protein.

In this regard, the present invention also provides a vector comprisinga DNA sequence, e.g., a Nostoc ellipsosporum gene sequence forcyanovirin, a cDNA encoding a cyanovirin, or a synthetic DNA sequenceencoding cyanovirin, a host cell comprising the vector, and a method ofusing such a host cell to produce a cyanovirin.

The DNA, whether isolated and purified or synthetic, or cDNA encoding acyanovirin can encode for either the entire cyanovirin or a portionthereof. Where the DNA or cDNA does not comprise the entire codingsequence of the native cyanovirin, the DNA or cDNA can be subcloned aspart of a gene fusion. In a transcriptional gene fusion, the DNA or cDNAwill contain its own control sequence directing appropriate productionof protein (e.g., ribosome binding site, translation initiation codon,etc.), and the transcriptional control sequences (e.g., promoterelements and/or enhancers) will be provided by the vector. In atranslational gene fusion, transcriptional control sequences as well asat least some of the translational control sequences (i.e., thetranslational initiation codon) will be provided by the vector. In thecase of a translational gene fusion, a chimeric protein will beproduced.

Genes also can be constructed for specific fusion proteins containing afunctional cyanovirin component plus a fusion component conferringadditional desired attribute(s) to the composite protein. For example, afusion sequence for a toxin or immunological reagent, as defined above,can be added to facilitate purification and analysis of the functionalprotein (e.g., such as the FLAG-cyanovirin-N fusion protein detailedwithin Examples 2-5).

Genes can be specifically constructed to code for fusion proteins, whichcontain a cyanovirin coupled to an effector protein, such as a toxin orimmunological reagent, for specific targeting to viral-infected, e.g.,HIV and/or HIV-infected, cells. In these instances, the cyanovirinmoiety serves not only as a neutralizing agent but also as a targetingagent to direct the effector activities of these molecules selectivelyagainst a given virus, such as HIV. Thus, for example, a therapeuticagent can be obtained by combining the HIV-targeting function of afunctional cyanovirin with a toxin aimed at neutralizing infectiousvirus and/or by destroying cells producing infectious virus, such asHIV. Similarly, a therapeutic agent can be obtained, which combines theviral-targeting function of a cyanovirin with the multivalency andeffector functions of various immunoglobulin subclasses.

Similar rationales underlie extensive developmental therapeutic effortsexploiting the HIV gp120-targeting properties of sCD4. For example,sCD4-toxin conjugates have been prepared in which sCD4 is coupled to aPseudomonas exotoxin component (Chaudhary et al., in The HumanRetrovirus, Gallo et al., eds., Academic Press: San Diego, 1991, pp.379-387; and Chaudhary et al., Nature 335, 369-372, 1988), or to adiphtheria toxin component (Aullo et al., EMBO J. 11, 575-583, 1992) orto a ricin A-chain component (Till et al., Science 242, 1166-1167,1988). Likewise, sCD4-immunoglobulin conjugates have been prepared inattempts to decrease the rate of in vivo clearance of functional sCD4activity, to enhance placental transfer, and to effect a targetedrecruitment of immunological mechanisms of pathogen elimination, such asphagocytic engulfment and killing by antibody-dependent cell-mediatedcytotoxicity, to kill and/or remove HIV-infected cells and virus (Caponet al., Nature 337, 525-531, 1989; Traunecker et al., Nature 339, 68-70,1989; and Langner et al., 1993, supra). While such CD4-immunoglobulinconjugates (sometimes called “immunoadhesins”) have, indeed, shownadvantageous pharmacokinetic and distributional attributes in vivo, andanti-HIV effects in vitro, clinical results have been discouraging(Schooley et al., 1990, supra; Husson et al., 1992, supra; and Langneret al., 1993, supra). This is not surprising since clinical isolates ofHIV, as opposed to laboratory strains, are highly resistant to bindingand neutralization by sCD4 (Orloff et al., 1995, supra; and Moore etal., 1992, supra). Therefore, the extraordinarily broad targetingproperties of a functional cyanovirin to viruses, e.g., primateretroviruses, in general, and clinical and laboratory strains, inparticular (Boyd et al., 1995, supra; and Gustafson et al., 1995,supra), can be especially advantageous for combining with toxins,immunoglobulins and other selected effector proteins.

Viral-targeted conjugates can be prepared either by genetic engineeringtechniques (see, for example, Chaudhary et al., 1988, supra) or bychemical coupling of the targeting component with an effector component.The most feasible or appropriate technique to be used to construct agiven cyanovirin conjugate or fusion protein will be selected based uponconsideration of the characteristics of the particular effector moleculeselected for coupling to a cyanovirin. For example, with a selectednon-proteinaceous effector molecule, chemical coupling, rather thangenetic engineering techniques, may be the only feasible option forcreating the desired cyanovirin conjugate.

Accordingly, the present invention also provides nucleic acid moleculesencoding cyanovirin fusion proteins. In particular, the presentinvention provides a nucleic acid molecule comprising SEQ ID NO:3 andsubstantially homologous sequences thereof. Also provided is a vectorcomprising a nucleic acid sequence encoding a cyanovirin fusion proteinand a method of obtaining a cyanovirin fusion protein by expression ofthe vector encoding a cyanovirin fusion protein in aprotein-synthesizing organism as described above. Accordingly,cyanovirin fusion proteins are also provided.

In view of the above, the present invention further provides an isolatedand purified nucleic acid molecule, which comprises a cyanovirin codingsequence, such as one of the aforementioned nucleic acids, namely anucleic acid molecule encoding an amino acid sequence of SEQ ID NO:2, anucleic acid molecule encoding an amino acid sequence of SEQ ID NO:4, anucleic acid molecule comprising a sequence of SEQ ID NO:1, or a nucleicacid molecule comprising a sequence of SEQ ID NO:3, coupled to a secondnucleic acid encoding an effector protein. The first nucleic acidpreferably comprises a nucleic acid sequence encoding at least ninecontiguous amino acids of the amino acid sequence of SEQ ID NO:2, whichencodes a functional cyanovirin, and the second nucleic acid preferablyencodes an effector protein, such as a toxin or immunological reagent asdescribed above.

Accordingly, the present invention also further provides an isolated andpurified protein encoded by a nucleic acid molecule comprising asequence of SEQ ID NO:1, a nucleic acid molecule comprising a sequenceof SEQ ID NO:3, a nucleic acid molecule encoding an amino acid sequenceof SEQ ID NO:2, or a nucleic acid molecule encoding an amino acidsequence of SEQ ID NO:4. Preferably, the aforementioned nucleic acidmolecules encode at least nine contiguous amino acids of the amino acidsequence of SEQ ID NO:2 coupled to an effector molecule, such as a toxinor immunological reagent as described above. Preferably, the effectormolecule targets a virus, more preferably HIV, and, most preferablyglycoprotein gp120. The coupling can be effected at the DNA level or bychemical coupling as described above. For example, a cyanovirin-effectorprotein conjugate of the present invention can be obtained by (a)selecting a desired effector protein or peptide; (b) synthesizing acomposite DNA coding sequence comprising a first DNA coding sequencecomprising one of the aforementioned nucleic acid sequences, which codesfor a functional cyanovirin, coupled to a second DNA coding sequence foran effector protein or peptide, e.g., a toxin or immunological reagent;(c) expressing said composite DNA coding sequence in an appropriateprotein-synthesizing organism; and (d) purifying the desired fusionprotein or peptide to substantially pure form. Alternatively, acyanovirin-effector molecule conjugate of the present invention can beobtained by (a) selecting a desired effector molecule and a cyanovirinor cyanovirin fusion protein; (b) chemically coupling the cyanovirin orcyanovirin fusion protein to the effector molecule; and (c) purifyingthe desired cyanovirin-effector molecule conjugate to substantially pureform.

Conjugates containing a functional cyanovirin coupled to a desiredeffector component, such as a toxin, immunological reagent, or otherfunctional reagent, can be designed even more specifically to exploitthe unique gp120-targeting properties of cyanovirins. Example 6 revealsnovel gp120-directed effects of cyanovirins. Additional insights weregained from solid-phase ELISA experiments (Boyd et al., 1995, supra).Both C-terminal gp120-epitope-specific capture or CD4-receptor captureof gp120, when detected either with polyclonal HIV-1-Ig or with mouseMAb to the immunodominant, third hypervariable (V3) epitope (Matsushitaet al., J. Virol. 62, 2107-2114, 1988), were strikingly inhibited bycyanovirin. Generally, engagement of the CD4 receptor does not interferewith antibody recognition of the V3 epitope, and vice versa (Moore etal., AIDS Res. Hum. Retrovir. 4, 369-379, 1988; and Matsushita et al.,1988, supra). However, cyanovirin apparently is capable of more globalconformational effects on gp120, as evidenced by loss ofimmunoreactivity at multiple, distinct, non-overlapping epitopes. Therange of antiviral activity (Boyd et al., 1995, supra) of cyanovirinagainst diverse CD4⁺-tropic immunodeficiency virus strains in varioustarget cells is remarkable; all tested strains of HIV-1, HIV-2 and SIVwere similarly sensitive to cyanovirin; clinical isolates and laboratorystrains showed essentially equivalent sensitivity. Cocultivation ofchronically infected and uninfected CEM-SS cells with cyanovirin did notinhibit viral replication, but did cause a concentration-dependentinhibition of cell-to-cell fusion and virus transmission; similarresults from binding and fusion inhibition assays employingHeLa-CD4-LTR-β-galactosidase cells were consistent with cyanovirininhibition of virus-cell and/or cell-cell binding.

The anti-viral, e.g., anti-HIV, activity of the cyanovirins andconjugates thereof of the present invention can be further demonstratedin a series of interrelated in vitro antiviral assays (Gulakowski etal., J. Virol. Methods 33, 87-100, 1991), which accurately predict forantiviral activity in humans. These assays measure the ability ofcompounds to prevent the replication of HIV and/or the cytopathiceffects of HIV on human target cells. These measurements directlycorrelate with the pathogenesis of HIV-induced disease in vivo. Theresults of the analysis of the antiviral activity of cyanovirins orconjugates, as set forth in Example 5 and as illustrated in FIGS. 5A-6D,are believed to predict accurately the antiviral activity of theseproducts in vivo in humans and, therefore, establish the utility of thepresent invention. Furthermore, since the present invention alsoprovides methods of ex vivo use of cyanovirins and conjugates (e.g., seeresults set forth in Example 5, and in FIGS. 5A-6D), the utility ofcyanovirins and conjugates thereof is even more certain.

The cyanovirins and conjugates thereof of the present invention can beshown to inhibit a virus, specifically a retrovirus, such as the humanimmunodeficiency virus, i.e., HIV-1 or HIV-2. The cyanovirins andconjugates of the present invention could be used to inhibit otherretroviruses as well as other viruses. Examples of viruses that may betreated in accordance with the present invention include, but are notlimited to, Type C and Type D retroviruses, HTLV-1, HTLV-2, HIV, FLV,SIV, MLV, BLV, BIV, equine infectious virus, anemia virus, avian sarcomaviruses, such as Rous sarcoma virus (RSV), hepatitis type A, B, non-Aand non-B viruses, arboviruses, varicella viruses, measles, mumps andrubella viruses.

Cyanovirins and conjugates thereof collectively comprise proteins andpeptides, and, as such, are particularly susceptible to hydrolysis ofamide bonds (e.g., catalyzed by peptidases) and disruption of essentialdisulfide bonds or formation of inactivating or unwanted disulfidelinkages (Carone et al., J. Lab. Clin. Med. 100, 1-14, 1982). There arevarious ways to alter molecular structure, if necessary, to provideenhanced stability to the cyanovirin or conjugate thereof (Wunsch,Biopolymers 22, 493-505, 1983; and Samanen, in Polymeric Materials inMedication, Gebelein et al., eds., Plenum Press: New York, 1985, pp.227-242), which may be essential for preparation and use ofpharmaceutical compositions containing cyanovirins or conjugates thereoffor therapeutic or prophylactic applications against viruses, e.g., HIV.Possible options for useful chemical modifications of a cyanovirin orconjugate include, but are not limited to, the following (adapted fromSamanen, J. M., 1985, supra): (a) olefin substitution, (b) carbonylreduction, (c) D-amino acid substitution, (d) N α-methyl substitution,(e) C α-methyl substitution, (f) C α-C′-methylene insertion, (g) dehydroamino acid insertion, (h) retro-inverso modification, (i) N-terminal toC-terminal cyclization, and (j) thiomethylene modification. Cyanovirinsand conjugates thereof also can be modified by covalent attachment ofcarbohydrate and polyoxyethylene derivatives, which are expected toenhance stability and resistance to proteolysis (Abuchowski et al., inEnzymes as Drugs, Holcenberg et al., eds., John Wiley: New York, 1981,pp. 367-378).

Other important general considerations for design of delivery strategysystems and compositions, and for routes of administration, for proteinand peptide drugs, such as cyanovirins and conjugates thereof (Eppstein,CRC Crit. Rev. Therapeutic Drug Carrier Systems 5, 99-139, 1988;Siddiqui et al., CRC Crit. Rev. Therapeutic Drug Carrier Systems 3,195-208, 1987); Banga et al., Int. J. Pharmaceutics 48, 15-50, 1988;Sanders, Eur. J. Drug Metab. Pharmacokinetics 15, 95-102, 1990; andVerhoef, Eur. J. Drug Metab. Pharmacokinetics 15, 83-93, 1990), alsoapply. The appropriate delivery system for a given cyanovirin orconjugate thereof will depend upon its particular nature, the particularclinical application, and the site of drug action. As with any proteinor peptide drug, oral delivery of a cyanovirin or a conjugate thereofwill likely present special problems, due primarily to instability inthe gastrointestinal tract and poor absorption and bioavailability ofintact, bioactive drug therefrom. Therefore, especially in the case oforal delivery, but also possibly in conjunction with other routes ofdelivery, it will be necessary to use an absorption-enhancing agent incombination with a given cyanovirin or conjugate thereof. A wide varietyof absorption-enhancing agents have been investigated and/or applied incombination with protein and peptide drugs for oral delivery and fordelivery by other routes (Verhoef, 1990, supra; van Hoogdalem, Pharmac.Ther. 44, 407-443, 1989; Davis, J. Pharm. Pharmacol. 44(Suppl. 1),186-190, 1992). Most commonly, typical enhancers fall into the generalcategories of (a) chelators, such as EDTA, salicylates, and N-acylderivatives of collagen, (b) surfactants, such as lauryl sulfate andpolyoxyethylene-9-lauryl ether, (c) bile salts, such as glycholate andtaurocholate, and derivatives, such as taurodihydrofusidate, (d) fattyacids, such as oleic acid and capric acid, and their derivatives, suchas acylcarnitines, monoglycerides and diglycerides, (e) non-surfactants,such as unsaturated cyclic ureas, (f) saponins, (g) cyclodextrins, and(h) phospholipids.

Other approaches to enhancing oral delivery of protein and peptidedrugs, such as the cyanovirins and conjugates thereof, can includeaforementioned chemical modifications to enhance stability togastrointestinal enzymes and/or increased lipophilicity. Alternatively,or in addition, the protein or peptide drug can be administered incombination with other drugs or substances, which directly inhibitproteases and/or other potential sources of enzymatic degradation ofproteins and peptides. Yet another alternative approach to prevent ordelay gastrointestinal absorption of protein or peptide drugs, such ascyanovirins or conjugates, is to incorporate them into a delivery systemthat is designed to protect the protein or peptide from contact with theproteolytic enzymes in the intestinal lumen and to release the intactprotein or peptide only upon reaching an area favorable for itsabsorption. A more specific example of this strategy is the use ofbiodegradable microcapsules or microspheres, both to protect vulnerabledrugs from degradation, as well as to effect a prolonged release ofactive drug (Deasy, in Microencapsulation and Related Processes,Swarbrick, ed., Marcell Dekker, Inc.: New York, 1984, pp. 1-60, 88-89,208-211). Microcapsules also can provide a useful way to effect aprolonged delivery of a protein and peptide drug, such as a cyanovirinor conjugate thereof, after injection (Maulding, J. Controlled Release6, 167-176, 1987).

Given the aforementioned potential complexities of successful oraldelivery of a protein or peptide drug, it is fortunate that there arenumerous other potential routes of delivery of a protein or peptidedrug, such as a cyanovirin or conjugate thereof. These routes includeintravenous, intraarterial, intrathecal, intracisternal, buccal, rectal,nasal, pulmonary, transdermal, vaginal, ocular, and the like (Eppstein,1988, supra; Siddiqui et al., 1987, supra; Banga et al., 1988, supra;Sanders, 1990, supra; Verhoef, 1990, supra; Barry, in Delivery Systemsfor Peptide Drugs, Davis et al., eds., Plenum Press: New York, 1986, pp.265-275; and Patton et al., Adv. Drug Delivery Rev. 8, 179-196, 1992).With any of these routes, or, indeed, with any other route ofadministration or application, a protein or peptide drug, such as acyanovirin or conjugate thereof, may initiate an immunogenic reaction.In such situations it may be necessary to modify the molecule in orderto mask immunogenic groups. It also can be possible to protect againstundesired immune responses by judicious choice of method of formulationand/or administration. For example, site-specific delivery can beemployed, as well as masking of recognition sites from the immune systemby use or attachment of a so-called tolerogen, such as polyethyleneglycol, dextran, albumin, and the like (Abuchowski et al., 1981, supra;Abuchowski et al., J. Biol. Chem. 252, 3578-3581, 1977; Lisi et al., J.Appl. Biochem. 4, 19-33, 1982; and Wileman et al., J. Pharm. Pharmacol.38, 264-271, 1986). Such modifications also can have advantageouseffects on stability and half-life both in vivo and ex vivo. Otherstrategies to avoid untoward immune reactions can also include theinduction of tolerance by administration initially of only low doses. Inany event, it will be apparent from the present disclosure to oneskilled in the art that for any particular desired medical applicationor use of a cyanovirin or conjugate thereof, the skilled artisan canselect from any of a wide variety of possible compositions, routes ofadministration, or sites of application, what is advantageous.

Accordingly, the antiviral cyanovirins and conjugates thereof of thepresent invention can be formulated into various compositions for useeither in therapeutic treatment methods for infected individuals, or inprophylactic methods against viral, e.g., HIV, infection of uninfectedindividuals.

The present invention also provides a pharmaceutical composition, whichcomprises an antiviral effective amount of an isolated and purifiedcyanovirin or cyanovirin conjugate and a pharmaceutically acceptablecarrier. The composition can further comprise an antiviral effectiveamount of at least one additional antiviral compound other than acyanovirin or conjugate thereof. Suitable antiviral compounds includeAZT, ddI, ddC, gancyclovir, fluorinated dideoxynucleosides, nevirapine,R82913, Ro 31-8959, BI-RJ-70, acyclovir, α-interferon, recombinant sCD4,michellamines, calanolides, nonoxynol-9, gossypol and derivativesthereof, and gramicidin. The cyanovirin used in the pharmaceuticalcomposition can be isolated and purified from nature or geneticallyengineered. Similarly, the cyanovirin conjugate can be geneticallyengineered or chemically coupled.

The present inventive compositions can be used to treat a virallyinfected animal, such as a human. The compositions of the presentinvention are particularly useful in inhibiting the growth orreplication of a virus, such as a retrovirus, in particular a humanimmunodeficiency virus, specifically HIV-1 and HIV-2. The compositionsare useful in the therapeutic or prophylactic treatment of animals, suchas humans, who are infected with a virus or who are at risk for viralinfection, respectively. The compositions also can be used to treatobjects or materials, such as medical equipment, supplies, or fluids,including biological fluids, such as blood, blood products, and tissues,to prevent viral infection of an animal, such as a human. Suchcompositions also are useful to prevent sexual transmission of viralinfections, e.g., HIV, which is the primary way in which the world'sAIDS cases are contracted (Merson, 1993, supra)

Potential virucides used or being considered for use against sexualtransmission of HIV are very limited; present agents in this categoryinclude nonoxynol-9 (Bird, AIDS 5, 791-796, 1991), gossypol andderivatives (Polsky et al., Contraception 39, 579-587, 1989; Lin,Antimicrob. Agents Chemother. 33, 2149-2151, 1989; and Royer, Pharmacol.Res. 24, 407-412, 1991), and gramicidin (Bourinbair, LifeSci./Pharmacol. Lett. 54, PL5-9, 1994; and Bourinbair et al.,Contraception 49, 131-137, 1994). The method of prevention of sexualtransmission of viral infection, e.g., HIV infection, in accordance withthe present invention comprises vaginal, rectal, oral, penile or othertopical treatment with an antiviral effective amount of a cyanovirinand/or cyanovirin conjugate, alone or in combination with anotherantiviral compound as described above.

Compositions for use in the prophylactic or therapeutic treatmentmethods of the present invention comprise one or more cyanovirin(s) orconjugate(s) thereof and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well-known to those who areskilled in the art, as are suitable methods of administration. Thechoice of carrier will be determined in part by the particularcyanovirin or conjugate thereof, as well as by the particular methodused to administer the composition.

One skilled in the art will appreciate that various routes ofadministering a drug are available, and, although more than one routemay be used to administer a particular drug, a particular route mayprovide a more immediate and more effective reaction than another route.Furthermore, one skilled in the art will appreciate that the particularpharmaceutical carrier employed will depend, in part, upon theparticular cyanovirin or conjugate thereof employed, and the chosenroute of administration. Accordingly, there is a wide variety ofsuitable formulations of the composition of the present invention.

Formulations suitable for oral administration can consist of liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or fruit juice; capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as solid or granules; solutions or suspensions in an aqueousliquid; and oil-in-water emulsions or water-in-oil emulsions. Tabletforms can include one or more of lactose, mannitol, corn starch, potatostarch, microcrystalline cellulose, acacia, gelatin, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid,and other excipients, colorants, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, and, pharmacologicallycompatible carriers. Suitable formulations for oral delivery can also beincorporated into synthetic and natural polymeric microspheres, or othermeans to protect the agents of the present invention from degradationwithin the gastrointestinal tract (see, for example, Wallace et al.,Science 260, 912-915, 1993).

The cyanovirins or conjugates thereof, alone or in combination withother antiviral compounds, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen and the like.

The cyanovirins or conjugates thereof, alone or in combinations withother antiviral compounds or absorption modulators, can be made intosuitable formulations for transdermal application and absorption(Wallace et al., 1993, supra). Transdermal electroporation oriontophoresis also can be used to promote and/or control the systemicdelivery of the compounds and/or compositions of the present inventionthrough the skin (e.g., see Theiss et al., Meth. Find. Exp. Clin.Pharmacol. 13, 353-359, 1991).

Formulations suitable for topical administration include lozengescomprising the active ingredient in a flavor, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbase, such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier; as well as creams, emulsions, gels and the like containing, inaddition to the active ingredient, such carriers as are known in theart.

Formulations for rectal administration can be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate. Formulations suitable for vaginal administration can bepresented as pessaries, tampons, creams, gels, pastes, foams, or sprayformulas containing, in addition to the active ingredient, such carriersas are known in the art to be appropriate. Similarly, the activeingredient can be combined with a lubricant as a coating on a condom.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

Formulations comprising a cyanovirin or cyanovirin conjugate suitablefor virucidal (e.g., HIV) sterilization of inanimate objects, such asmedical supplies or equipment, laboratory equipment and supplies,instruments, devices, and the like, can, for example, be selected oradapted as appropriate, by one skilled in the art, from any of theaforementioned compositions or formulations. Preferably, the cyanovirinis produced by recombinant DNA technology. The cyanovirin conjugate canbe produced by recombinant DNA technology or by chemical coupling of acyanovirin with an effector molecule as described above. Similarly,formulations suitable for ex vivo virucidal sterilization of blood,blood products, sperm, or other bodily products or tissues, or any othersolution, suspension, emulsion or any other material which can beadministered to a patient in a medical procedure, can be selected oradapted as appropriate by one skilled in the art, from any of theaforementioned compositions or formulations. However, suitableformulations for such ex vivo applications or for virucidal treatment ofinanimate objects are by no means limited to any of the aforementionedformulations or compositions. One skilled in the art will appreciatethat a suitable or appropriate formulation can be selected, adapted ordeveloped based upon the particular application at hand.

For ex vivo uses, such as virucidal treatments of inanimate objects ormaterials, blood or blood products, or tissues, the amount ofcyanovirin, or conjugate or composition thereof, to be employed shouldbe sufficient that any virus or virus-producing cells present will berendered noninfectious or will be destroyed. For example, for HIV, thiswould require that the virus and/or the virus-producing cells be exposedto concentrations of cyanovirin-N in the range of 0.1-1000 nM. Similarconsiderations apply to in vivo applications. Therefore, the designationof “antiviral effective amount” is used generally to describe the amountof a particular cyanovirin, conjugate or composition thereof requiredfor antiviral efficacy in any given application.

For in vivo uses, the dose of a cyanovirin, or conjugate or compositionthereof, administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a prophylacticor therapeutic response in the individual over a reasonable time frame.The dose used to achieve a desired antiviral concentration in vivo(e.g., 0.1-1000 nM) will be determined by the potency of the particularcyanovirin or conjugate employed, the severity of the disease state ofinfected individuals, as well as, in the case of systemicadministration, the body weight and age of the infected individual. Thesize of the dose also will be determined by the existence of any adverseside effects that may accompany the particular cyanovirin, or conjugateor composition thereof, employed. It is always desirable, wheneverpossible, to keep adverse side effects to a minimum.

The dosage can be in unit dosage form, such as a tablet or capsule. Theterm “unit dosage form” as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of a cyanovirin or conjugatethereof, alone or in combination with other antiviral agents, calculatedin an amount sufficient to produce the desired effect in associationwith a pharmaceutically acceptable diluent, carrier, or vehicle.

The specifications for the unit dosage forms of the present inventiondepend on the particular cyanovirin, or conjugate or compositionthereof, employed and the effect to be achieved, as well as thepharmacodynamics associated with each cyanovirin, or conjugate orcomposition thereof, in the host. The dose administered should be an“antiviral effective amount” or an amount necessary to achieve an“effective level” in the individual patient.

Since the “effective level” is used as the preferred endpoint fordosing, the actual dose and schedule can vary, depending uponinterindividual differences in pharmacokinetics, drug distribution, andmetabolism. The “effective level” can be defined, for example, as theblood or tissue level (e.g., 0.1-1000 nM) desired in the patient thatcorresponds to a concentration of one or more cyanovirin or conjugatethereof, which inhibits a virus, such as HIV, in an assay known topredict for clinical antiviral activity of chemical compounds andbiological agents. The “effective level” for agents of the presentinvention also can vary when the cyanovirin, or conjugate or compositionthereof, is used in combination with AZT or other known antiviralcompounds or combinations thereof.

One skilled in the art can easily determine the appropriate dose,schedule, and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired “effectiveconcentration” in the individual patient. One skilled in the art alsocan readily determine and use an appropriate indicator of the “effectorconcentration” of the compounds of the present invention by a direct(e.g., analytical chemical analysis) or indirect (e.g., with surrogateindicators such as p24 or RT) analysis of appropriate patient samples(e.g., blood and/or tissues).

In the treatment of some virally infected individuals, it can bedesirable to utilize a “mega-dosing” regimen, wherein a large dose ofthe cyanovirin or conjugate thereof is administered, time is allowed forthe drug to act, and then a suitable reagent is administered to theindividual to inactivate the drug.

The pharmaceutical composition can contain other pharmaceuticals, inconjunction with the cyanovirin or conjugate thereof, when used totherapeutically treat a viral infection, such as that which results inAIDS. Representative examples of these additional pharmaceuticalsinclude antiviral compounds, virucides, immunomodulators,immunostimulants, antibiotics and absorption enhancers. Exemplaryantiviral compounds include AZT, ddI, ddC, gancylclovir, fluorinateddideoxynucleosides, nonnucleoside analog compounds, such as nevirapine(Shih et al., PNAS 88, 9878-9882, 1991), TIBO derivatives, such asR82913 (White et al., Antiviral Res. 16, 257-266, 1991), BI-RJ-70(Merigan, Am. J. Med. 90 (Suppl. 4A), 8S-17S, 1991), michellamines (Boydet al., J. Med. Chem. 37, 1740-1745, 1994) and calanolides (Kashman etal., J. Med. Chem. 351 2735-2743, 1992), nonoxynol-9, gossypol andderivatives, and gramicidin (Bourinbair et al., 1994, supra). Exemplaryimmunomodulators and immunostimulants include various interleukins,sCD4, cytokines, antibody preparations, blood transfusions, and celltransfusions. Exemplary antibiotics include antifungal agents,antibacterial agents, and anti-Pneumocystitis carnii agents. Exemplaryabsorption enhancers include bile salts and other surfactants, saponins,cyclodextrins, and phospholipids (Davis, 1992, supra).

Administration of a cyanovirin or conjugate thereof with otheranti-retroviral agents and particularly with known RT inhibitors, suchas ddC, AZT, ddI, ddA, or other inhibitors that act against other HIVproteins, such as anti-TAT agents, is expected to inhibit most or allreplicative stages of the viral life cycle. The dosages of ddC and AZTused in AIDS or ARC patients have been published. A virustatic range ofddC is generally between 0.05 μM to 1.0 μM. A range of about 0.005-0.25mg/kg body weight is virustatic in most patients. The preliminary doseranges for oral administration are somewhat broader, for example 0.001to 0.25 mg/kg given in one or more doses at intervals of 2, 4, 6, 8, 12,etc. hours. Currently, 0.01 mg/kg body weight ddC given every 8 hrs ispreferred. When given in combined therapy, the other antiviral compound,for example, can be given at the same time as the cyanovirin orconjugate thereof or the dosing can be staggered as desired. The twodrugs also can be combined in a composition. Doses of each can be lesswhen used in combination than when either is used alone.

It will also be appreciated by one skilled in the art that a DNAsequence of a cyanovirin or conjugate thereof of the present inventioncan be inserted ex vivo into mammalian cells previously removed from agiven animal, in particular a human, host. Such cells can be employed toexpress the corresponding cyanovirin or conjugate in vivo afterreintroduction into the host. Feasibility of such a therapeutic strategyto deliver a therapeutic amount of an agent in close proximity to thedesired target cells and pathogens, i.e., virus, more particularlyretrovirus, specifically HIV and its envelope glycoprotein gp120, hasbeen demonstrated in studies with cells engineered ex vivo to expresssCD4 (Morgan et al., 1994, supra). It is also possible that, as analternative to ex vivo insertion of the DNA sequences of the presentinvention, such sequences can be inserted into cells directly in vivo,such as by use of an appropriate viral vector. Such cells transfected invivo are expected to produce antiviral amounts of cyanovirin or aconjugate thereof directly in vivo.

The present inventive cyanovirins, conjugates, compositions and methodsare further described in the context of the following examples. Theseexamples serve to illustrate further the present invention and are notintended to limit the scope of the invention.

EXAMPLES Example 1

This example shows details of anti-HIV bioassay-guided isolation andelucidation of pure cyanovirin from aqueous extracts of the culturedcyanobacterium, Nostoc ellipsosporum.

The method described in Weislow et al. (1989, supra) was used to monitorand direct the isolation and purification process. Cyanobacterialculture conditions, media and classification were as describedpreviously, (Patterson, J. Phycol. 27, 530-536, 1991). Briefly, thecellular mass from a unialgal strain of Nostoc ellipsosporum (cultureQ68D170) was harvested by filtration, freeze-dried and extracted withMeOH—CH₂Cl₂ (1:1) followed by H₂O. Bioassay indicated that only the H₂Oextract contained HIV-inhibitory activity. A solution of the aqueousextract (30 mg/ml) was treated by addition of an equal volume of ethanol(EtOH). The resulting 1:1 H₂O—EtOR solution was kept at −20° C. for 15hrs. Then, the solution was centrifuged to remove precipitated materials(presumably, high molecular weight biopolymers). The resultingHIV-inhibitory supernatant was evaporated, then fractionated byreverse-phase vacuum-liquid chromatography (Coll et al., J. Nat. Prod.49, 934-936, 1986; and Pelletier et al., J. Nat. Prod. 49, 892-900,1986) on wide-pore C₄ packing (300 Å, BakerBond WP-C₄), and eluted withincreasing concentrations of methanol (MeOH) in H₂O. Anti-HIV activitywas concentrated in the material eluted with MeOH—H₂O (2:1). SDS-PAGEanalysis of this fraction showed one main protein band, with a relativemolecular mass (Mr) of approximately 10 kDa. Final purification wasachieved by repeated reverse-phase HPLC on 1.9×15 cm μBondapak C₁₈(Waters Associates) columns eluted with a gradient of increasingconcentration of acetonitrile in H₂O. The mobile phase contained 0.05%(v/v) TFA, pH=2. Eluted proteins and peptides were detected by UVabsorption at 206, 280 and 294 nm with a rapid spectral detector(Pharmacia LKB model 2140). Individual fractions were collected, pooledbased on the UV chromatogram, and lyophilized. Pooled HPLC fractionswere subjected to SDS-PAGE under reducing conditions (Laemmli, Nature227, 680-685, 1970), conventional amino acid analysis, and testing foranti-HIV activity.

FIG. 1A is a graph of OD 206 nm versus time (min), which shows theμBondapak C₁₈ HPLC chromatogram of nonreduced cyanovirin eluted with alinear CH3CN/H₂O gradient (buffered with 0.05% TFA) from 28-38% CH₃CN.FIG. 1C is a graph of OD 206 nm versus time (min), which shows thechromatogram of cyanovirin that was first reduced with β-mercaptoethanoland then separated under identical HPLC conditions. HPLC fractions fromthe two runs were collected as indicated. 10% aliquots of each fractionwere lyophilized, made up in 100 μl 3:1 H₂O/DMSO and assessed foranti-HIV activity in the XTT assay. FIG. 1B is a bar graph of maximumdilution for 50% protection versus HPLC fraction, which illustrates themaximum dilution of each fraction that provided 50% protection from thecytopathic effects of HIV infection for the nonreduced cyanovirin HPLCfractions. Corresponding anti-HIV results for the HPLC fractions fromreduced cyanovirin are shown in FIG. 1D, which is a bar graph of maximumdilution for 50% protection versus HPLC fraction. 20% aliquots ofselected HPLC fractions were analyzed by SDS-PAGE.

In the initial HPLC separation, using a linear gradient from 30-50%CH₃CN, the anti-HIV activity coeluted with the principal UV-absorbingpeak at approximately 33% CH₃CN. Fractions corresponding to the activepeak were pooled and split into two aliquots.

Reinjection of the first aliquot under similar HPLC conditions, but witha linear gradient from 28-38% CH₃CN, resolved the active material intotwo closely eluting peaks at 33.4 and 34.0% CH₃CN. The anti-HIV activityprofile of the fractions collected during this HPLC run (as shown inFIG. 1B) corresponded with the two UV peaks (as shown in FIG. 1A).SDS-PAGE of fractions collected under the individual peaks showed only asingle protein band.

The second aliquot from the original HPLC separation was reduced withβ-mercaptoethanol prior to reinjection on the HPLC. Using an identical28-38% gradient, the reduced material gave one principal peak (as shownin FIG. 1C) that eluted later in the run with 36.8% CH₃CN. Only a traceof anti-HIV activity was detected in the HPLC fractions from the reducedmaterial (as shown in FIG. 1D).

The two closely eluting HPLC peaks of the nonreduced material (FIG. 1A)gave only one identical band on SDS-PAGE (run under reducing conditions)and reduction with β-mercaptoethanol resulted in an HPLC peak with alonger retention time than either of the nonreduced peaks. Thisindicated that disulfides were present in the native protein. Amino acidanalysis of the two active peaks showed they had virtually identicalcompositions. It is possible that the two HPLC peaks resulted fromcis/trans isomerism about a proline residue or from microheterogeneityin the protein sample that was not detected in either the amino acidanalysis or during sequencing. The material collected as the twoHIV-inhibitory peaks was combined for further analyses and was given thename cyanovirin-N.

Example 2

This example illustrates synthesis of cyanovirin genes.

The chemically deduced amino acid sequence of cyanovirin-N wasback-translated to obtain a DNA coding sequence. In order to facilitateinitial production and purification of recombinant cyanovirin-N, acommercial expression vector (pFLAG-1, from InternationalBiotechnologies, Inc., New Haven, Conn.), for which reagents wereavailable for affinity purification and detection, was selected.Appropriate restriction sites for ligation to pFLAG-1, and a stop codon,were included in the DNA sequence. FIG. 2 is an example of a DNAsequence encoding a synthetic cyanovirin gene. This DNA sequence designcouples the cyanovirin-N coding region to codons for a “FLAG”octapeptide at the N-terminal end of cyanovirin, providing forproduction of a FLAG-cyanovirin fusion protein.

A flowchart for synthesis of this DNA sequence is depicted in FIG. 9.

The DNA sequence was synthesized as 13 overlapping, complementaryoligonucleotides and assembled to form the double-stranded codingsequence. Oligonucleotide elements of the synthetic DNA coding sequencewere synthesized using a dual-column nucleic acid synthesizer (Model392, Applied Biosystems Inc., Foster City, Calif.). Completedoligonucleotides were cleaved from the columns and deprotected byincubation overnight at 56° C. in concentrated ammonium hydroxide. Priorto treatment with T4 polynucleotide kinase, 33-66 mers weredrop-dialyzed against distilled water. The 13 oligonucleotidepreparations were individually purified by HPLC, and 10 mole quantitiesof each were ligated with T4 DNA ligase into a 327 bp double-strandedDNA sequence. DNA was recovered and purified from the reaction buffer byphenol:chloroform extraction, ethanol precipitation, and further washingwith ethanol. Individual oligonucleotide preparations were pooled andboiled for 10 min to ensure denaturation. The temperature of the mixturewas then reduced to 70° C. for annealing of the complementary strands.After 20 min, the tube was cooled on ice and 2,000 units of T4 DNAligase were added together with additional ligase buffer. Ligation wasperformed overnight at 16° C. DNA was recovered and purified from theligation reaction mixture by phenol:chloroform extraction and ethanolprecipitation and washing.

The purified, double-stranded synthetic DNA was then used as a templatein a polymerase chain reaction (PCR). One μl of the DNA solutionobtained after purification of the ligation reaction mixture was used asa template. Thermal cycling was performed using a Perkin-Elmerinstrument. “Vent” thermostable DNA polymerase, restriction enzymes, T4DNA ligase and polynucleotide kinase were obtained from New EnglandBiolabs, Beverly, Mass. Vent polymerase was selected for thisapplication because of its claimed superiority in fidelity compared tothe usual Taq enzyme. The PCR reaction product was run on a 2% agarosegel in TBE buffer. The 327 bp construct was then cut from the gel andpurified by electroelution. Because it was found to be relativelyresistant to digestion with Hind III and Xho I restriction enzymes, itwas initially cloned using the pCR-Script system (Stratagene). Digestionof a plasmid preparation from one of these clones yielded the codingsequence, which was then ligated into the multicloning site of thepFLAG-1 vector.

E. coli were transformed with the pFLAG-construct and recombinant cloneswere identified by analysis of restriction digests of plasmid DNA.Sequence analysis of one of these selected clones indicated that fourbases deviated from the intended coding sequence. This included deletionof three bases coding for one of four cysteine residues contained in theprotein and an alteration of the third base in the preceding codon(indicated by the boxes in FIG. 2). In order to correct these“mutations,” which presumably arose during the PCR amplification of thesynthetic template, a double-stranded “patch” was synthesized, whichcould be ligated into restriction sites flanking the mutations (theseBst XI and Esp1 sites are also indicated in FIG. 2). The patch wasapplied and the repair was confirmed by DNA sequence analysis.

For preparation of a DNA sequence coding for native cyanovirin, theaforementioned FLAG-cyanovirin construct was subjected to site-directedmutagenesis to eliminate the codons for the FLAG octapeptide and, at thesame time, to eliminate a unique Hind III restriction site. Thisprocedure is illustrated in FIG. 3, which illustrates a site-directedmutagenesis maneuver used to eliminate codons for a FLAG octapeptide anda Hind III restriction site from the sequence of FIG. 2. A mutagenicoligonucleotide primer was synthesized, which included portions of thecodons for the Omp secretory peptide and cyanovirin, but lacking thecodons for the FLAG peptide. Annealing of this mutagenic primer, withcreation of a DNA hairpin in the template strand, and extension by DNApolymerase resulted in generation of new plasmid DNA lacking both theFLAG codon sequence and the Hind III site (refer to FIG. 2 for details).Digestion of plasmid DNA with Hind III resulted in linearization of“wild-type” strands but not “mutant” strands. Since transformation of E.coli occurs more efficiently with circular DNA, clones could be readilyselected which had the revised coding sequence which specifiedproduction of native cyanovirin-N directly behind the Omp secretorypeptide. DNA sequencing verified the presence of the intended sequence.Site-directed mutagenesis reactions were carried out using materials(polymerase, buffers, etc.) obtained from Pharmacia Biotech, Inc.,Piscataway, N.J.

Example 3

This example illustrates expression of synthetic cyanovirin genes.

As depicted in FIG. 10:

E. coli (strain DH5α) were transformed (by electroporation) with thepFLAG-1 vector containing the coding sequence for the FLAG-cyanovirin-Nfusion protein (see FIG. 2 for details of the DNA sequence). Selectedclones were seeded into small-scale shake flasks containing (LB) growthmedium with 100 μg/ml ampicillin and expanded by incubation at 37° C.Larger-scale Erlenmeyer flasks (0.5-3.0 liters) were then seeded andallowed to grow to a density of 0.5-0.7 OD₆₀₀ units. Expression of theFLAG-cyanovirin-N fusion protein was then induced by adding IPTG to afinal concentration of 1.7 mM and continuing incubation at 30° C. for3-6 hrs. For harvesting of periplasmic proteins, bacteria were pelleted,washed, and then osmotically shocked by treatment with sucrose, followedby resuspension in distilled water. Periplasmic proteins were obtainedby sedimenting the bacteria and then filtering the aqueous supernatantthrough Whatman paper. Crude periplasmic extracts showed both anti-HIVactivity and presence of a FLAG-cyanovirin-N fusion protein by Westernor spot-blotting.

The construct for native cyanovirin-N described in Example 2 was used totransform bacteria in the same manner as described above for theFLAG-cyanovirin-N fusion protein. Cloning, expansion, induction withIPTG, and harvesting were performed similarly. Crude periplasmicextracts showed strong anti-HIV activity on bioassay.

Example 4

This example illustrates purification of recombinant cyanovirinproteins.

Using an affinity column based on an anti-FLAG monoclonal antibody(International Biotechnologies, Inc., New Haven, Conn.),FLAG-cyanovirin-N fusion protein could be purified as depicted in FIG.11:

The respective periplasmic extract, prepared as described in Example 3,was loaded onto 2-20 ml gravity columns containing affinity matrix andwashed extensively with PBS containing CA⁺⁺ to remove contaminatingproteins. Since the binding of the FLAG peptide to the antibody isCa⁺⁺-dependent, fusion protein could be eluted by passage of EDTAthrough the column. Column fractions and wash volumes were monitored byspot-blot analysis using the same anti-FLAG antibody. Fractionscontaining fusion protein were then pooled, dialyzed extensively againstdistilled water, and lyophilized.

For purification of recombinant native cyanovirin-N, the correspondingperiplasmic extract from Example 3 was subjected to step-gradient C₄reverse-phase, vacuum-liquid chromatography to give three fractions: (1)eluted with 100% H₂O, (2) eluted with MeOH—H₂O (2:1), and (3) elutedwith 100% MeOH. The anti-HIV activity was concentrated in fraction (2).Purification of the recombinant cyanovirin-N was performed by HPLC on a1.9×15 cm μBondapak (Waters Associates) C₁₈ column eluted with agradient of increasing concentration of CH₃CN in H₂O (0.05% TFA, v/v inthe mobile phase). A chromatogram of the final HPLC purification on a1×10 cm (Cohensive Technologies, Inc.) C₄ column monitored at 280 nm isshown in FIG. 4, which is typical HPLC chromatogram during thepurification of a recombinant native cyanovirin. Gradient elution, 5ml/min, from 100% H₂O to H₂O—CH₃CN (7:3) was carried out over 23 minwith 0.05% TFA (v/v) in the mobile phase.

Example 5

This example shows anti-HIV activities of natural and recombinantcyanovirin-N and FLAG-cyanovirin-N.

Pure proteins were initially evaluated for antiviral activity using anXTT-tetrazolium anti-HIV assay described previously (Boyd, in AIDS,Etiology, Diagnosis, Treatment and Prevention, 1988, supra; Gustafson etal., J. Med. Chem. 35, 1978-1986, 1992; Weislow, 1989, supra; andGulakowski, 1991, supra). The CEM-SS human lymphocytic target cell lineused in all assays was maintained in RPMI 1650 medium (Gibco, GrandIsland, N.Y.), without phenol red, and was supplemented with 5 fetalbovine serum, 2 mM L-glutamine, and 50 μg/ml gentamicin (completemedium).

Exponentially growing cells were pelleted and resuspended at aconcentration of 2.0×10⁵ cells/ml in complete medium. The Haitianvariant of HIV, HTLV-III_(RF) (3.54×10⁶ SFU/ml), was used throughout.Frozen virus stock solutions were thawed immediately before use andresuspended in complete medium to yield 1.2×10⁵ SFU/ml. The appropriateamounts of the pure proteins for anti-HIV evaluations were dissolved inH₂O—DMSO (3:1), then diluted in complete medium to the desired initialconcentration. All serial drug dilutions, reagent additions, andplate-to-plate transfers were carried out with an automated Biomek 1000Workstation (Beckman Instruments, Palo Alto, Calif.).

FIGS. 5A-5C are graphs of a control versus concentration (nm), whichillustrate antiviral activities of native cyanovirin from Nostocellipsosporum (A), recombinant native (B), and recombinant FLAG-fusion(C) cyanovirins. The graphs show the effects of a range ofconcentrations of the respective cyanovirins upon CEM-SS cells infectedwith HIV-1 (), as determined after 6 days in culture. Data pointsrepresent the percent of the respective uninfected, nondrug-treatedcontrol values. All three cyanovirins showed potent anti-HIV activity,with an EC₅₀ in the low, nanomolar range and no significant evidence ofdirect cytotoxicity to the host cells at the highest testedconcentrations (up to 1.2 μM).

As an example of a further demonstration of the anti-HIV activity ofpure cyanovirin-N, a battery of interrelated anti-HIV assays wasperformed in individual wells of 96-well microtiter plates, usingmethods described in detail elsewhere (Gulakowski, 1991, supra).Briefly, the procedure was as follows. Cyanovirin solutions wereserially diluted in complete medium and added to 96-well test plates.Uninfected CEM-SS cells were plated at a density of 1×10⁴ cells in 50 μlof complete medium. Diluted HIV-1 was then added to appropriate wells ina volume of 50 μl to yield a multiplicity of infection of 0.6.Appropriate cell, virus, and drug controls were incorporated in eachexperiment. The final volume in each microtiter well was 200 μl.Quadruplicate wells were used for virus-infected cells. Plates wereincubated at 37° C. in an atmosphere containing 5% CO₂ for 4, 5, or 6days.

Subsequently, aliquots of cell-free supernatant were removed from eachwell using the Biomek, and analyzed for reverse transcriptase activity,p24 antigen production, and synthesis of infectious virions as described(Gulakowski, 1991, supra). Cellular growth or viability then wasestimated on the remaining contents of each well using the XTT (Weislowet al., 1989, supra), BCECF (Rink et al., J. Cell Biol. 95, 189-196,1982), and DAPI (McCaffrey et al., In Vitro Cell Develop. Biol. 24,247-252, 1988) assays as described (Gulakowski et al., 1991, supra). Tofacilitate graphical displays and comparisons of data, the individualexperimental assay results (of at least quadruplicate determinations ofeach) were averaged, and the mean values were used to calculatepercentages in reference to the appropriate controls. Standard errors ofthe mean values used in these calculations typically averaged less than10% of the respective mean values.

FIGS. 6A-6D are graphs of % control versus concentration (nm), whichillustrate anti-HIV activity of a cyanovirin in a multiparameter assayformat. Graphs 6A, 6B, and 6C show the effects of a range ofconcentrations of cyanovirin upon uninfected CEM-SS cells (∘), and uponCEM-SS cells infected with HIV-1 (), as determined after 6 days inculture. Graph 6A depicts the relative numbers of viable CEM-SS cells,as assessed by the BCECF assay. Graph 6B depicts the relative DNAcontents of the respective cultures. Graph 6C depicts the relativenumbers of viable CEM-SS cells, as assessed by the XTT assay. Graph 6Dshows the effects of a range of concentrations of cyanovirin uponindices of infectious virus or viral replication as determined after 4days in culture. These indices include viral reverse transcriptase (▴),viral core protein p24 (♦), and syncytium-forming units (▪). In graphs6A, 6B, and 6C, the data are represented as the percent of theuninfected, nondrug-treated control values. In graph 6D the data arerepresented as the percent of the infected, nondrug-treated controlvalues.

As illustrated in FIG. 6, cyanovirin-N was capable of completeinhibition of the cytopathic effects of HIV-1 upon CEM-SS humanlymphoblastoid target cells in vitro; direct cytotoxicity of the proteinupon the target cells was not observed at the highest testedconcentrations. Cyanovirin-N also strikingly inhibited the production ofRT, p24, and SFU in HIV-1-infected CEM-SS cells within these sameinhibitory effective concentrations, indicating that the protein haltedviral replication.

The anti-HIV activity of the cyanovirins is extremely resilient to harshenvironmental challenges. For example, unbuffered cyanovirin-N solutionswithstood repeated freeze-thaw cycles or dissolution in organic solvents(up to 100% DMSO, MeOH, or CH₃CN) with no loss of activity. Cyanovirin-Ntolerated detergent (0.1% SDS), high salt (6 M guanidine HCl) and heattreatment (boiling, 10 min in H₂O) with no significant loss ofHIV-inhibitory activity. Reduction of the disulfides withβ-mercaptoethanol, followed immediately by C₁₈ HPLC purification,drastically reduced the cytoprotective activity of cyanovirin-N.However, solutions of reduced cyanovirin-N regained anti-HIV inhibitoryactivity during prolonged storage. When cyanovirin-N was reduced(β-mercaptoethanol, 6M guanidine HCl pH 8.0) but not put through C₁₈HPLC, and, instead, simply desalted, reconstituted and assayed, itretained virtually full activity.

Example 6

This example illustrates that the HIV viral envelope gp120 is aprincipal molecular target of cyanovirin-N.

Initial experiments, employing the XTT-tetrazolium assay (Weislow etal., 1989, supra), revealed that host cells preincubated with cyanovirin(10 nM, 1 hr), then centrifuged free of cyanovirin-N, retained normalsusceptibility to HIV infection; in contrast, the infectivity ofconcentrated virus similarly pretreated, then diluted to yieldnon-inhibitory concentrations of cyanovirin-N, was essentiallyabolished. This indicated that cyanovirin-N was acting directly upon thevirus itself, i.e., acting as a direct “virucidal” agent to preventviral infectivity even before it could enter the host cells. This wasfurther confirmed in time-of-addition experiments, likewise employingthe XTT-tetrazolium assay (Weislow, 1989, supra), which showed that, toafford maximum antiviral activity, cyanovirin-N had to be added to cellsbefore or as soon as possible after addition of virus as shown in FIG.7, which is a graph of % uninfected control versus time of addition(hrs), which shows results of time-of-addition studies of a cyanovirin,showing anti-HIV activity in CEM-SS cells infected with HIV-1_(RF).Introduction of cyanovirin () or ddC (▪) (10 nM and 5 μMconcentrations, respectively) was delayed by various times after initialincubation, followed by 6 days incubation, then assay of cellularviability (FIG. 7A) and RT (open bars, FIG. 7B). Points representaverages (±S.D.) of at least triplicate determinations. In markedcontrast to the reverse transcriptase inhibitor ddC, delay of additionof cyanovirin-N by only 3 hrs resulted in little or no antiviralactivity (FIG. 7B). The aforementioned results suggested thatcyanovirin-N inhibited HIV-infectivity by interruption of the initialinteraction of the virus with the cell; this would, therefore, likelyinvolve a direct interaction of cyanovirin-N with the viral gp120. Thiswas confirmed by ultrafiltration experiments and dot-blot assays.

Ultrafiltration experiments were performed to determine if soluble gp120and cyanovirin-N could bind directly, as assessed by inhibition ofpassage of cyanovirin-N through a 50 kDa-cutoff ultrafilter. Solutionsof cyanovirin (30 μg) in PBS were treated with various concentrations ofgp120 for 1 hr at 37° C., then filtered through a 50 kDa-cutoffcentrifugal ultrafilter (Amicon). After washing 3 times with PBS,filtrates were desalted with 3 kDa ultrafilters; retentates werelyophilized, reconstituted in 100 μl H₂O and assayed for anti-HIVactivity.

FIG. 8 is a graph of OD (450 nm) versus cyanovirin concentration(μg/ml), which illustrates cyanovirin/gp120 interactions defining gp120as a principal molecular target of cyanovirin. Free cyanovirin-N wasreadily eluted, as evidenced by complete recovery of cyanovirin-Nbioactivity in the filtrate. In contrast, filtrates from cyanovirin-Nsolutions treated with gp120 revealed a concentration-dependent loss offiltrate bioactivity; moreover, the 50 kDa filter retentates were allinactive, indicating that cyanovirin-N and soluble gp120 interacteddirectly to form a complex incapable of binding to gp120 of intactvirus.

There was further evidence of a direct interaction of cyanovirin-N andgp120 in a PVDF membrane dot-blot assay. A PVDF membrane was spottedwith 5 μg CD4 (CD), 10 μg aprotinin (AP), 10 μg bovine globulin (BG),and decreasing amounts of cyanovirin; 6 μg [1], 3 μg [2], 1.5 μg [3],0.75 μg [4], 0.38 μg [5], 0.19 μg [6], 0.09 μg [7], and 0.05 μg [8],then washed with PBST and visualized per manufacturer's recommendations.FIG. 8 dot blot of binding of cyanovirin and a gp120-HRP conjugate(Invitrogen), showed that cyanovirin-N specifically bound a horseradishperoxidase conjugate of gp120 (gp120-HRP) in a concentration-dependentmanner.

All of the references cited herein are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred compounds and methods may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the following claims.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 4(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 327 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:          (A) NAME/KEY: CDS           (B) LOCATION: 10..312    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1:CGATCGAAG CTT GGT AAA TTC TCC CAG ACC TGC TAC# AAC TCC GCT ATC         48           Leu Gly Lys Phe Ser #Gln Thr Cys Tyr Asn Ser Ala Ile             1      #         5          #         10CAG GGT TCC GTT CTG ACC TCC ACC TGC GAA CG#T ACC AAC GGT GGT TAC       96Gln Gly Ser Val Leu Thr Ser Thr Cys Glu Ar #g Thr Asn Gly Gly Tyr     15              #     20              #     25AAC ACC TCC TCC ATC GAC CTG AAC TCC GTT AT#C GAA AAC GTT GAC GGT      144Asn Thr Ser Ser Ile Asp Leu Asn Ser Val Il #e Glu Asn Val Asp Gly 30                  # 35                  # 40                  # 45TCC CTG AAA TGG CAG CCG TCC AAC TTC ATC GA#A ACC TGC CGT AAC ACC      192Ser Leu Lys Trp Gln Pro Ser Asn Phe Ile Gl #u Thr Cys Arg Asn Thr                 50  #                 55  #                 60CAG CTG GCT GGT TCC TCC GAA CTG GCT GCT GA#A TGC AAA ACC CGT GCT      240Gln Leu Ala Gly Ser Ser Glu Leu Ala Ala Gl #u Cys Lys Thr Arg Ala             65      #             70      #             75CAG CAG TTC GTT TCC ACC AAA ATC AAC CTG GA#C GAC CAC ATC GCT AAC      288Gln Gln Phe Val Ser Thr Lys Ile Asn Leu As #p Asp His Ile Ala Asn         80          #         85          #         90ATC GAC GGT ACC CTG AAA TAC GAA TAACTCGAGA TC #GTA                 #    327 Ile Asp Gly Thr Leu Lys Tyr Glu      95              #    100(2) INFORMATION FOR SEQ ID NO: 2:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 101 amino  #acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:Leu Gly Lys Phe Ser Gln Thr Cys Tyr Asn Se #r Ala Ile Gln Gly Ser  1               5  #                 10  #                 15Val Leu Thr Ser Thr Cys Glu Arg Thr Asn Gl #y Gly Tyr Asn Thr Ser             20      #             25      #             30Ser Ile Asp Leu Asn Ser Val Ile Glu Asn Va #l Asp Gly Ser Leu Lys         35          #         40          #         45Trp Gln Pro Ser Asn Phe Ile Glu Thr Cys Ar #g Asn Thr Gln Leu Ala     50              #     55              #     60Gly Ser Ser Glu Leu Ala Ala Glu Cys Lys Th #r Arg Ala Gln Gln Phe 65                  # 70                  # 75                  # 80Val Ser Thr Lys Ile Asn Leu Asp Asp His Il #e Ala Asn Ile Asp Gly                 85  #                 90  #                 95Thr Leu Lys Tyr Glu             100 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 327 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:          (A) NAME/KEY: CDS           (B) LOCATION: 1..327    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:GAC TAC AAG GAC GAC GAT GAC AAG CTT GGT AA#A TTC TCC CAG ACC TGC       48Asp Tyr Lys Asp Asp Asp Asp Lys Leu Gly Ly #s Phe Ser Gln Thr Cys  1               5  #                 10  #                 15TAC AAC TCC GCT ATC CAG GGT TCC GTT CTG AC#C TCC ACC TGC GAA CGT       96Tyr Asn Ser Ala Ile Gln Gly Ser Val Leu Th #r Ser Thr Cys Glu Arg             20      #             25      #             30ACC AAC GGT GGT TAC AAC ACC TCC TCC ATC GA#C CTG AAC TCC GTT ATC      144Thr Asn Gly Gly Tyr Asn Thr Ser Ser Ile As #p Leu Asn Ser Val Ile         35          #         40          #         45GAA AAC GTT GAC GGT TCC CTG AAA TGG CAG CC#G TCC AAC TTC ATC GAA      192Glu Asn Val Asp Gly Ser Leu Lys Trp Gln Pr #o Ser Asn Phe Ile Glu     50              #     55              #     60ACC TGC CGT AAC ACC CAG CTG GCT GGT TCC TC#C GAA CTG GCT GCT GAA      240Thr Cys Arg Asn Thr Gln Leu Ala Gly Ser Se #r Glu Leu Ala Ala Glu 65                  # 70                  # 75                  # 80TGC AAA ACC CGT GCT CAG CAG TTC GTT TCC AC#C AAA ATC AAC CTG GAC      288Cys Lys Thr Arg Ala Gln Gln Phe Val Ser Th #r Lys Ile Asn Leu Asp                 85  #                 90  #                 95GAC CAC ATC GCT AAC ATC GAC GGT ACC CTG AA #A TAC GAA              #    327 Asp His Ile Ala Asn Ile Asp Gly Thr Leu Ly #s Tyr Glu            100       #           105 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 109 amino #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4:Asp Tyr Lys Asp Asp Asp Asp Lys Leu Gly Ly #s Phe Ser Gln Thr Cys  1               5  #                 10  #                 15Tyr Asn Ser Ala Ile Gln Gly Ser Val Leu Th #r Ser Thr Cys Glu Arg             20      #             25      #             30Thr Asn Gly Gly Tyr Asn Thr Ser Ser Ile As #p Leu Asn Ser Val Ile         35          #         40          #         45Glu Asn Val Asp Gly Ser Leu Lys Trp Gln Pr #o Ser Asn Phe Ile Glu     50              #     55              #     60Thr Cys Arg Asn Thr Gln Leu Ala Gly Ser Se #r Glu Leu Ala Ala Glu 65                  # 70                  # 75                  # 80Cys Lys Thr Arg Ala Gln Gln Phe Val Ser Th #r Lys Ile Asn Leu Asp                 85  #                 90  #                 95Asp His Ile Ala Asn Ile Asp Gly Thr Leu Ly #s Tyr Glu            100       #           105

What is claimed is:
 1. A method of inhibiting binding of an envelopedvirus to a cell in a host, which method comprises administering to thehost an antiviral effective amount of an isolated and purified antiviralprotein having an amino acid sequence of SEQ ID NO: 2, whereuponadministration of said antiviral effective amount of said antiviralprotein, binding of the enveloped virus to the cell is inhibited.